Fluvoxamine is a more potent inhibitor of lidocaine metabolism than ketoconazole and erythromycin in vitro

CYP3A4 is generally believed to be the major CYP enzyme involved in the biotransformation of lidocaine in man; however, recent in vivo studies suggest that this may not be the case. We have examined the effects of the CYP3A4 inhibitors erythromycin and ketoconazole and the CYP1A2 inhibitor fluvoxamine on the N-deethylation, i.e. formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine by human liver microsomes. The experiments were carried out at lidocaine concentrations of 5 microM (clinically relevant concentration) and 800 microM. The formation of both MEGX and 3-hydroxylidocaine was best described by a two-enzyme model. At 5 microM of lidocaine, fluvoxamine was a potent inhibitor of the formation of MEGX (IC50 1.2 microM). Ketoconazole and erythromycin also showed an inhibitory effect on MEGX formation, but ketoconazole (IC50 8.5 microM) was a much more potent inhibitor than erythromycin (IC50 200 microM). At 800 microM of lidocaine, fluvoxamine (IC50 20.7 microM) and ketoconazole (IC50 20.4 microM) displayed a modest inhibitory effect on MEGX formation, whereas erythromycin was a weak inhibitor (IC50 >250 microM). The 3-hydroxylation of lidocaine was potently inhibited by fluvoxamine at both lidocaine concentrations (IC50 0.16 microM at 5 microM and 1.8 microM at 800 microM). Erythromycin and ketoconazole showed a clear inhibitory effect on the 3-hydroxylation of lidocaine at 5 microM of lidocaine (IC50 9.9 microM and 13.9 microM, respectively), but did not show a consistent effect at 800 microM of lidocaine (IC50 >250 microM and 75.0 microM, respectively). Although further studies are needed to elucidate the role of distinct CYP enzymes in the biotransformation of lidocaine in humans, the findings of this study suggest that while both CYP1A2 and CYP3A4 are involved in the metabolism of lidocaine by human liver microsomes, CYP1A2 is the more important isoform at clinically relevant lidocaine concentrations.     

Fluvoxamine is a Potent Inhibitor of the Metabolism of Caffeine in vitro

Abstract: The selective serotonin re-uptake inhibitor, fluvoxamine, is a very potent inhibitor of CYP1A2, and accordingly causes pharmacokinetic interactions with drugs metabolised by CYP1A2, such as caffeine, theophylline, imipramine, tacrine and clozapine. Interaction between caffeine and fluvoxamine has been described in vivo, leading to lowering of total clearance of caffeine by 80% during fluvoxamine intake. The main purpose of the present study was to evaluate this interaction in vitro in human liver microsomes. A high-performance liquid chromatography method was developed in order to assay 1, 3-dimethylxanthine, 1, 7-dimethylxanthine, 3, 7-dimethylxanthine and 1, 3, 7-trimethyluric acid formed from caffeine by human liver microsomes. The limit of detection was 0.06 nmol · mg protein^?1 · hr^?1. As expected, fluvoxamine was a very potent inhibitor of the formation of the N-demethylated caffeine metabolites, displaying K_i values of 0.08–0.28 μM. The formation of 1, 7-dimethylxanthine was virtually abolished by 10 μM of fluvoxamine, indicating that the N3-demethylation of caffeine is almost exclusively catalysed by CYP1A2. The CYP3A4 inhibitors, ketoconazole and bromocriptine, inhibited 1, 3, 7-trimethyluric acid formation with K s of 0.75 μM and 5 μM, respectively, thus further supporting the involvement of CYP3A4 in the 8-hydroxylation of caffeine. The study shows that fluvoxamine, as expected, is a potent inhibitor of the metabolism of caffeine in vitro.     

Involvement of CYP1A2 and CYP3A4 in lidocaine N-deethylation and 3-hydroxylation in humans.

The roles of cytochrome P-450 (CYP) enzymes in the N-deethylation, i.e., formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine were studied with human liver microsomes and recombinant human CYP isoforms. Both CYP1A2 and CYP3A4 were found to be capable of catalyzing the formation of MEGX and 3-OH-lidocaine. Lidocaine N-deethylation by liver microsomes was strongly inhibited by furafylline (by about 60%) and anti-CYP1A1/2 antibodies (>75%) at 5 microM lidocaine, suggesting that CYP1A2 was the major isoform catalyzing lidocaine N-deethylation at low (therapeutically relevant) lidocaine concentrations. Troleandomycin inhibited the N-deethylation of lidocaine by about 50% at 800 microM lidocaine, suggesting that the role of CYP3A4 may be more important than that of CYP1A2 at high lidocaine concentrations. Chemical inhibition and immunoinhibition studies also indicated that 3-OH-lidocaine formation was catalyzed almost exclusively by CYP1A2, CYP3A4 playing only a minor role. Although the CYP2C9 inhibitor sulfaphenazole (100 microM) inhibited MEGX formation by about 30%, recombinant human CYP2C9 showed very low catalytic activity, suggesting a negligible role for this enzyme in lidocaine N-deethylation. Chemical inhibition studies indicated that CYP2C19, CYP2D6, and CYP2E1 did not play significant roles in the metabolism of lidocaine in vitro. Taken together, these results demonstrate that CYP1A2 and CYP3A4 enzymes are the major CYP isoforms involved in lidocaine N-deethylation. Therefore, the MEGX test (formation of MEGX from lidocaine) is not a suitable marker of hepatic CYP3A4 activity in vivo.     

Variation in the Response of Clozapine Biotransformation Pathways in Human Hepatic Microsomes to CYP1A2‐ and CYP3A4‐selective Inhibitors

The atypical antipsychotic agent clozapine (CLZ) is effective in many patients who are resistant to conventional antipsychotic drugs. Cytochromes P450 (CYPs) 1A2 and 3A4 oxidize CLZ to norCLZ and CLZ N‐oxide in human liver. Concurrent treatment with inducers and inhibitors of CYP1A2 modulates CLZ elimination that disrupts therapy. Drug–drug interactions involving CYP3A4 are also significant but less predictable. To further characterize the factors underlying these interactions, we used samples from a cohort of human livers to assess variation in CLZ oxidation pathways in relation to intrinsic CYP3A4 and CYP1A2 activities and the effects of the corresponding selective inhibitors ketoconazole (0.2 and 2 μM) and fluvoxamine (1 and 10 μM). The CYP3A4‐selective inhibitor ketoconazole (2 μM) impaired CLZ N‐oxide formation in all 14 of the livers used in inhibition studies (≥50% inhibition) while the CYP1A2‐selective inhibitor fluvoxamine (10 μM) decreased norCLZ formation in nine. Ketoconazole effectively inhibited CLZ metabolism in five of seven livers that catalysed CYP3A4‐dependent testosterone 6β‐hydroxylation at or above the median rate and in four other livers with lower intrinsic CYP3A4 activity. Similarly, fluvoxamine (10 μM) readily inhibited CLZ oxidation in seven livers with high CYP1A2‐mediated 7‐ethoxyresorufin O‐deethylation activity (at or above the median) and three livers with lower intrinsic CYP1A2 activity. In three livers, CLZ biotransformation was impaired by both ketoconazole and fluvoxamine, consistent with a major role for both CYPs. These findings suggest that the intrinsic activities of CYPs 1A2 and 3A4 are unrelated to the response to CYP‐selective inhibitors and that assessment of the activities in vivo may not assist the prediction of drug–drug interactions.     

Midazolam α-Hydroxylation by Human Liver Microsomes in vitro: Inhibition by Calcium Channel Blockers,Itraconazole and Ketoconazole

Abstract: The inhibitory effects of five calcium channel blockers (diltiazem, isradipine, mibefradil, nifedipine and verapamil) and three azole antifungal agents (itraconazole, hydroxyitraconazole and ketoconazole) on the α-hydroxylation of midazolam, a probe drug for CYP3A4-mediated interactions in humans, were studied in vitro using human liver microsomes. IC₅₀ and K_i values were determined for each inhibitor. The kinetics of the formation of α-hydroxymidazolam were best described by simple Michaelis-Menten kinetics. The estimated values of V_max and K_m were 696 pmol min.^?1 mg^?1 and 7.46 μmol l^?1, respectively. All the compounds studied inhibited midazolam α-hydroxylation activity in a concentration-dependent manner, but there were marked differences in their relative inhibitory potency. Ketoconazole was the most potent inhibitor of midazolam α-hydroxylation (IC₅₀ 0.12 μmol l^?1), being 10 times more potent than itraconazole (IC₅₀ 1.2 μmol l^?1). The inhibitory effect of hydroxyitraconazole (IC₅₀ 2.3 μmol l^?1) was almost as large as that of itraconazole. Among the calcium channel blockers, mibefradil was the most potent inhibitor of the α-hydroxylation of midazolam, with an IC₅₀ value (1.6 μmol l^?1) similar to that of itraconazole. The other calcium channel blockers were much weaker inhibitors than mibefradil: verapamil exhibited a modest inhibitory effect with an IC₅₀ of 23 μmol l^?1, while isradipine, nifedipine and diltiazem, with IC₅₀ values ranging from 57 to >100 μmol l^?1, were weak inhibitors. This rank order of potency against the α-hydroxylation of midazolam was verified by the K_i values. With the exception of diltiazem, these in vitro results conform with the observed interaction potential of these agents with midazolam and many other CYP3A4 substrates in vivo in man.     

Effect of erythromycin and rifampicin on monoethylglycinexylidide test

Background:

The dynamic liver function test based on the hepatic conversion of lidocaine to monoethylglycinexylidide (MEGX) provides a direct measure of the actual functional state of the liver. Cytochrome P450 (CYP) 3A4 has been proposed as the main CYP isoform responsible for MEGX formation. The concomitant use of either CYP3A4 inducer rifampicin or CYP3A4 inhibitor erythromycin may influence the results of MEGX test. Hence, the objective of this study was to evaluate the effect of a CYP3A4 inhibitor erythromycin and inducer rifampicin on the MEGX test.

Materials and Methods:

The study included 20 healthy male volunteers whose routine laboratory tests were normal. As per study protocol, MEGX test was carried out in all the participants after an overnight fast. All the participants were given 1 mg/kg lidocaine dose intravenously and MEGX concentration at 30 and 60 min after IV dose was measured using HPLC. These MEGX values served as control values. Ten subjects received 600 mg/day erythromycin orally for six days while remaining ten participants received 600 mg/day rifampicin orally for six days. On the sixth day, MEGX test was carried out two hours after the last dose.

Result:

Rifampicin increased the mean plasma concentration of MEGX₃₀ from 93.94 ± 26.31 to 98.54 ± 24.94 μg/ml (P = 0.085) and MEGX₆₀ from 134.34 ± 35.42 to 136.36 ± 33.14 μg/ml (P = 0.051). Erythromycin lowered the serum concentration of MEGX₃₀ from 101.37 ± 39.39 to 96.67 ± 36.09 μg/ml (P = 0.128) and MEGX₆₀ from 142.52 ± 42.65 to 138.98 ± 40.23 μg/ml (P = 0.159).

Conclusion:

It can be concluded from this study that the MEGX test is not affected by concomitant administration of CYP3A4 modifiers rifampicin and erythromycin.     

Inhibitory Effects of Silibinin on Cytochrome P‐450 Enzymes in Human Liver Microsomes

Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P‐450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7–300 μM) at 37° in 0.1 M KH₂PO₄ buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC₅₀ values were determined for each substrate at respective K_M concentrations. Silibinin had little effect (IC₅₀>200 μM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)‐mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC₅₀=173 μM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC₅₀=29 μM and 46 μM) and S(?)‐warfarin 7‐hydroxylation (CYP2C9; IC₅₀=43 μM and 45 μM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (K_i,c=2.3 μM and 2.4 μM). Inhibition was competitive for S(?)‐warfarin 7‐hydroxylation (K_i,c=18 μM and 19 μM) and mainly non‐competitive for denitronifedipine oxidation (K_i,n=9 μM and 12 μM). With therapeutic silibinin peak plasma concentrations of 0.6 μM and biliary concentrations up to 200 μM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.     

Cooperativity of α-naphthoflavone in cytochrome P450 3A-dependent drug oxidation activities in hepatic and intestinal microsomes from mouse and human

1. The effects of several CYP3A substrates (α-naphthoflavone (αNF), terfenadine, midazolam, erythromycin) on nifedipine oxidation and testosterone 6-β-hydroxylation activities were investigated in hepatic and intestinal microsomes from mouse and human. 2. αNF (10 μM) and terfenadine (100 μM) inhibited nifedipine oxidation activities (at substrate concentration of 100 μM) in mouse hepatic microsomes to ～50%, but not in mouse intestinal microsomes. αNF (30 μM) stimulated nifedipine oxidation activities in mouse and human intestinal microsomes and in human hepatic microsomes to ～1.3-1.8-fold. Inhibitory potencies (50% inhibition concentration, IC₅₀) of midazolam and erythromycin for nifedipine oxidations were calculated to be ～90 μM in human intestinal microsomes. In contrast, testosterone (100 μM) stimulated the nifedipine oxidation activities ～1.5-fold in hepatic and intestinal microsomes from mouse and human. 3. αNF showed different effects on the kinetic parameters including the Hill coefficients of nifedipine oxidation and testosterone 6-β-hydroxylation catalysed by hepatic and intestinal microsomes from mouse and human. Cooperativity in nifedipine oxidation was increased by the addition of αNF to pooled human hepatic microsomes, but little effects of αNF could be observed in individual human intestinal microsomes. 4. These results suggest that CYP3A enzymes in liver and intestine might have different characteristics and that observations from hepatic microsomes should not be directly applicable to intestine metabolism in some cases. Studies of drug-drug interactions of CYP3A substrates are recommended to be performed using intestinal samples.     

Inhibition of CYP3A4 and CYP3A5 catalyzed metabolism of alprazolam and quinine by ketoconazole as racemate and four different enantiomers

Objective The antifungal drug ketoconazole (KTZ) is known as an inhibitor of, especially, the CYP3A subfamily, which catalyzes the metabolism of a large variety of drugs. Interactions between KTZ and CYP3A substrates have been reported both in vivo and in vitro. Most of them, however, involved the KTZ racemate. KTZ racemate and the separate enantiomers, 2R,4R; 2R,4S; 2S,4S, and 2S,4R, were evaluated for their selectivity in inhibiting alprazolam and quinine metabolism. Methods The inhibition of alprazolam and quinine metabolism was studied in an in vitro system of human liver microsomes (HLM), recombinant of CYP3A4 and CYP3A5. The concentrations of formed 3-hydroxyquinine and 4- and α-hydroxyalprazolam were measured by HPLC and LC-MS, respectively. Results Quinine 3-hydroxylation was catalyzed to a similar extent by CYP3A4 and CYP3A5. The formation rate of 4-hydroxyalprazolam was higher than that of α-hydroxyalprazolam for each HLM, CYP3A4 and CYP3A5. KTZ racemate and enantiomers showed differential inhibitory effects of quinine and alprazolam metabolism. Quinine metabolism catalyzed by HLM, CYP3A4 and CYP3A5 was potently inhibited by the trans-enantiomer KTZ 2S,4S, with IC₅₀ value of 0.16 μM for HLM, 0.04 μM for CYP3A4 and 0.11 μM for CYP3A5. The same enantiomer showed the lowest IC₅₀ values of 0.11 μM for HLM and 0.04 μM for CYP3A5 with respect to alprazoalm 4-hydroxylation and also the same pattern for alprazolamα-hydroxylation, 0.13 μM for HLM and 0.05 μM for CYP3A5. Alprazolam metabolism (both α- and 4- hydroxylations) catalyzed by CYP3A4 was inhibited potently by the cis-enantiomer KTZ 2S,4R, with IC₅₀ values of 0.03 μM . Conclusions Alprazolam and quinine metabolism is catalyzed by both CYP3A4 and CYP3A5. The present study showed that different KTZ enantiomers inhibit CYP3A4 and CYP3A5 to different degrees, indicating that structural differences among the enantiomers would be related to their inhibitory potency on these two enzymes.     

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1′-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC₅₀ = 0.76 μM (29), K_i = 0.68 μM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC₅₀ declining to 0.32 μM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were k_inact = 0.024 min^?1 (30) and K_I = 1.63 μM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC₅₀ and K_i values of 0.057 μM (17) and 0.043 μM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).     

Inhibitory Effects of Trospium Chloride on Cytochrome P450 Enzymes in Human Liver Microsomes

Abstract Trospium chloride, an atropine derivative used for the treatment of urge incontinence, was tested for inhibitory effects on human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using the following selective substrates: dextromethorphan (CYP2D6), denitronifedipine (CYP3A4), caffeine (CYP1A2), chlorzoxazone (CYP2E1), S-(+)-mephenytoin (CYP2C19), S-(-)-warfarin (CYP2C9) and coumarin (CYP2A6). Incubations with each substrate were carried out without a possible inhibitor and in the presence of trospium chloride at varying concentrations (37–3000 μM) at 37° in 0.1 M KH₂PO₄ buffer containing up to 3% DMSO. Metabolite concentrations were determined by high-performance liquid chromatography (HPLC) in all cases except CYP2A6 where direct fluorescence spectroscopy was used. First, trospium chloride IC₅₀ values were determined for each substrate at respective K_M concentrations. Trospium chloride did not show relevant inhibitory effects on the metabolism of most substrates (IC₅₀ values considerably higher than 1 mM). The only clear inhibition was seen for the CYP2D6-dependent high-affinity O-demethylation of dextromethorphan, where IC₅₀ values of 27 μM and 44 μM were observed. Therefore, additional dextromethorphan concentrations (0.4–2000 μM) were tested. Trospium chloride was a competitive inhibitor of the reaction with K_i values of 20 and 51 μM, respectively. Thus, trospium chloride has negligible inhibitory effects on CYP3A4, CYP1A2, CYP2E1, CYP2C19, CYP2C9 and CYP2A6 activity but is a reasonably potent inhibitor of CYP2D6 in vitro. Compared to therapeutic trospium chloride peak plasma concentrations below 50 nM, the 1000-times higher competitive inhibition constant K_i however suggests that inhibition of CYP2D6 by trospium chloride is without any clinical relevance.     

Selegiline Metabolism and Cytochrome P450 Enzymes:In vitro Study in Human Liver Microsomes*

Although being a drug therapeutically used for a long time, the enzymatic metabolism of selegiline has not been adequately studied. In the current work we have studied the cytochrome P450 (CYP)‐catalyzed oxidative metabolism of selegiline to desmethylselegiline and l‐methamphetamine and the effects of selegiline, desmethylselegiline and l‐methamphetamine on hepatic CYP enzymes in human liver microsomes in vitro. The apparent K_m values for desmethylselegiline and l‐methamphetamine formation were on an average 149 μM and 293 μM, and the apparent V_max values, 243 pmol/min./mg and 1351 pmol/min./mg, respectively. Furafylline and ketoconazole, the known reference inhibitors for CYP1A2 and CYP3A4, respectively, inhibited the formation of desmethylselegiline with K_i value of 1.7 μM and 15 μM. Ketoconazole inhibited also the formation of l‐methamphetamine with K_i of 18 μM. Fluvoxamine, an inhibitor of CYP1A2, CYP2C19 and CYP3A4, inhibited the formation of desmethylselegiline and l‐methamphetamine with K_i values of 9 and 25 μM, respectively. On the basis of these results we suggest that CYP1A2 and CYP3A4 contribute to the formation of desmethylselegiline and that CYP3A4 participates in the formation of l‐methamphetamine. In studies with CYP‐specific model activities, both selegiline and desmethylselegiline inhibited the CYP2C19‐mediated S‐mephenytoin 4′‐hydroxylation with average IC₅₀ values of 21 μM and 26 μM, respectively. The K_i for selegiline was determined to be around 7 μM. Selegiline inhibited CYP1A2‐mediated ethoxyresorufin O‐deethylation with a K_i value of 76 μM. Inhibitory potencies of selegiline, desmethylselegiline and l‐methamphetamine towards other CYP‐model activities were much lower. On this basis, selegiline and desmethylselegiline were shown to have a relatively high affinity for CYP2C19, but no evidence about selegiline metabolism by CYP2C19 was obtained.     

Pronounced differences in inhibition potency of lactone and non-lactone compounds for mouse and human coumarin 7-hydroxylases (CYP2A5 and CYP2A6)

1. The structural requirements for a compound to be a potent inhibitor for mouse CYP2A5 and human CYP2A6 enzymes catalysing coumarin 7-hydroxylase activity have been studied. 2. The IC₅₀ of 28 compounds for the pyrazole-treated male DBA/2 mouse and human liver microsomal coumarin 7-hydroxylation were determined at 10 muM coumarin concentration 15 times over K_m of coumarin. 3. The three most potent inhibitors for CYP2A5 were gamma-nonanoic lactone, gamma-decanolactone and gamma-phenyl-gamma-butyrolactone with an IC₅₀=1.9 +/- 0.4, 2.1 +/- 0.2 and 2.4 +/- 0.3 muM and for CYP2A6 7-methylcoumarin, butylcyclohexane and indan with an IC₅₀=30 +/- 3.2, 43 +/- 9 and 50 +/- 11 muM. 4. Among the 28 compounds studied, only 2-benzoxazolinone, 2-indanone and gamma- valerolactone showed similar inhibitory activity in both species. Indan had a lower IC₅₀ for human than for mouse coumarin 7-hydroxylation, whereas the IC₅₀ of 24 other compounds was higher for human than for mouse coumarin 7-hydroxylation. 5. The largest difference in IC₅₀ between mouse and human activity was observed with 5-substituted phenyl, pentyl, hexyl, heptyl or octyl gamma-lactones or 6-substituted delta-lactones. IC₅₀ of gamma-undecanolactone and gamma-decanolactone was 500 times lower for mouse than human coumarin 7-hydroxylation. 6. The difference in the IC₅₀ between human and mouse coumarin 7-hydroxylation decreased substantially with the corresponding compounds without the lactone ring. 7. It is concluded that certain 5- or 6-position substituted gamma- and delta -lactones are potent inhibitors for mouse CYP2A5 but not for the orthologous human CYP2A6 and that the active site of CYP2A6 could be smaller than the active site of CYP2A5.     

Metabolism of Dextromethorphan in vitro: Involvement of Cytochromes P450 2D6 AND 3A3/4, with a Possible Role of 2E1

Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O-demethylation to dextrorphan (DOP), and N-demethylation to 3-methoxy-morphinan (MEM), in humans. 3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4. DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting CYP2D6 activity (mean K_i for quinidine, 0·1 ± 0·13 μM). Kinetic parameters for the two O-demethylation mediating enzymes predict an average relative intrinsic clearance (V_max/K_m ratio) of 96% of total O-demethylation mediated via the high-affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor CYP2D6 activity. The Eadie–Hofstee plot of DMO N-demethylation was consistent with single-enzyme Michaelis–Menten kinetics (V_max varying from 3·3 to 6·8 nmol mg⁻¹ min⁻¹, K_m from 231 to 322 μM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean K_i an order of magnitude higher (0·37 μM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC₅₀ varying from 1 to 2·6 μM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 μM between 33 and 43%. Chemical inhibitors of CYP2D6 (quinidine), CYP1A1/2 (α-naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo. © 1997 John Wiley & Sons, Ltd.     

Identification of human cytochrome P450 enzymes involved in the formation of 4-hydroxyestazolam from estazolam

To predict drug interactions with estazolam, the biotransformation of estazolam to its major hydoxylated metabolite, 4-hydroxyestazolam was studied in vitro using pooled human liver microsomes and individual expressed human cytochrome P450 (CYP) enzymes. Estazolam was metabolized to 4-hydroxyestazolam according to the Hill kinetic model in pooled human liver microsomes. The K_m value for the 4-hydroxylation of estazolam was 24.1?µM, and the V_max value was 52.6?pmol?min^?1?mg^?1 protein. The formation of 4-hydroxyestazolam from estazolam in pooled human liver microsomes was significantly inhibited by itraconazole and erythromycin, specific CYP3A4 inhibitors, in a dose-dependent manner, with IC₅₀ values of 1.1 and 12.8?µM, respectively. When estazolam was incubated with expressed human CYP enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4), it was metabolized only by CYP3A4. In conclusion, the biotransformation of estazolam to 4-hydroxyestazolam was catalyzed by CYP3A4.     

Comparative 1-Substituted Imidazole Inhibition of Cytochrome P450 Isozyme-Selective Activities in Human and Mouse Hepatic Microsomes

Inhibition of cytochrome P450(CYP)-selective reactions in a single human and a single mouse hepatic microsome preparation by fourteen 1-substituted imidazoles provides a simultaneous ranking of reaction susceptibility to a specific imidazole and the relative inhibitory potency of the imidazoles for a given reaction. CYP3A4/5 activity was inhibited (IC₅₀ <5 μM) by the greatest number of imidazoles, followed closely by CYP2C9. Seven imidazoles exhibited IC₅₀ values for CYP3A4/5 <0.3 μM (none for CYP2C9) and were exclusively above 300 MW. Nafimidone (MW, 236) exhibited an IC₅₀ value <0.3 μM towards CYP2D6 and CYP1A2 reactions. CYP2E1 and CYP2A6 were exclusively inhibited (IC₅₀ <5 μM) by imidazoles with MWs below ～200. In general, mouse activities exhibited lower IC₅₀ values than in human microsomes.     

Assessment of specificity of eight chemical inhibitors using cDNA-expressed cytochromes P450

1. The selectivity of eight chemical inhibitors has been extensively evaluated with 10 cDNA-expressed human cytochrome P450 isoforms (CYP). The results indicate that sulphaphenazole, quinidine and α-naphthoflavone are selective inhibitors of CYP2C9 (IC₅₀ = 5 0.5-0.7 μM), CYP2D6 (0.3-0.4 μM) and CYP1A (0.05-5 μM) respectively on the basis of the IC₅₀, which are much lower than those of other P450 isoforms (> 10-fold). 2. Ketoconazole exhibited potent inhibition of both CYP3A4-catalysed metabolism of phenanthrene, testosterone, diazepam (IC₅₀ = 0.03-0.5 μM) and CYP1A1-catalysed deethylation of 7-ethoxycoumarin (0.33 μM). The selectivity of ketoconazole for other P450s was highly related to the concentration used. 3. Diethyldithiocarbamate, orphenadrine and furafylline were shown separately to be less selective inhibitors of CYP2E1, CYP2B6 and CYP1A isoforms by a broad range of IC₅₀ that overlap those observed with other P450 isoforms. 4. Furafylline, quinidine and α-naphthoflavone activated CYP3A4-catalysed phenanthrene metabolism by 1.7-, 2- and 15-fold respectively. 5. The selectivity of orphenadrine and ketoconazole was further examined by using inhibitory monoclonal antibodies (MAb). Inhibitory MAb specific for the individual P450 isoforms may be of greater value than chemical inhibitors.     

Enantiospecific effects of chiral drugs on cytochrome P450 inhibition in vitro

1.?The aim of this work was to examine the differences in the inhibitory potency of individual enantiomers and racemic mixtures of selected chiral drugs on human liver microsomal cytochromes P450.

2.?The interaction of enantiomeric forms of six drugs (tamsulosin, tolterodine, citalopram, modafinil, zopiclone, ketoconazole) with nine cytochromes P450 (CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1A2) was examined. HPLC methods were used to estimate the extent of the inhibition of specific activity in vitro.

3.?Tamsulosin (TAM) and tolterodine (TOL) inhibited CYP3A4 activity with an enantiospecific pattern. The inhibition of CYP3A4 activity differed for R-TAM (K_i 2.88?±?0.12?µM) and S-TAM (K_i 14.22?±?0.53?µM) as well as for S-TOL (K_i 1.71?±?0.03?µM) and R-TOL (K_i 4.78?±?0.17?µM). Also, the inhibition of CYP2C19 by ketoconazole (KET) cis-enantiomers exhibited enantioselective behavior: the (+)-KET (IC₅₀ 23.64?±?6.25?µM) was more potent than (?)-KET (IC₅₀ 66.12?±?12.6?µM). The inhibition of CYP2C19 by modafinil (MOD) enantiomers (R-MOD IC₅₀?=?51.79?±?8.58?µM, S-MOD IC₅₀?=?48.62?±?9.74?µM) and the inhibition of CYP2D6 by citalopram (CIT) enantiomers (R-CIT IC₅₀?=?68.17?±?5.70?µM, S-CIT IC₅₀?=?62.63?±?7.89?µM) was not enantiospecific.

4.?Although enantiospecific interactions were found (TAM, TOL, KET), they are probably not clinically relevant as the plasma levels are generally lower than the drug concentration needed for prominent inhibition (at least 50% of CYP activity).     

Fluvoxamine inhibits the CYP2C19-catalysed metabolism of proguanil in vitro

Objective: The potent CYP1A2 inhibitor fluvoxamine has recently been shown also to be an effective inhibitor of the CYP2C19-mediated metabolism of the antimalarial drug proguanil in vivo. The purpose of the present study was to confirm this interaction in vitro. Methods: A high-performance liquid chromatography (HPLC) method was developed to assay 4-chlorophenylbiguanide (4-CPBG) and cycloguanil formed from proguanil by microsomes prepared from human liver. The limit of detection was 0.08?nmol?·?mg^?1?·?h^?1. Results: The formation of 4-CPBG and cycloguanil could be described by one-enzyme kinetics, indicating that the formation of the two metabolites is almost exclusively catalysed by a single enzyme, i.e. CYP2C19 within the concentration range used, or that the contribution of an alternative low-affinity enzyme, probably CYP3A4, is very low. This notion was confirmed by the lack of potent inhibition by four CYP3A4 inhibitors: ketoconazole, bromocriptine, midazolam and dihydroergotamine. Fluvoxamine was a very effective inhibitor of the oxidation of proguanil, displaying K_i values of 0.69?μmol?·?l^?1 for the inhibition of cycloguanil formation and 4.7?μmol?·?l^?1 for the inhibition of 4-CPBG formation. As expected, the CYP2C19 substrate omeprazole inhibited the formation of both metabolites with an IC₅₀ of 10?μmol?·?l^?1. Norfluoxetine and sulfaphenazole inhibited proguanil oxidation with K_i values of 7.3–16?μmol?·?l^?1, suggesting that the two compounds are moderate inhibitors of CYP2C19. Conclusions: Fluvoxamine is a fairly potent inhibitor of CYP2C19 and it has the potential for causing drug-drug interactions with substrates for CYP2C19 such as imipramine, clomipramine, amitriptyline and diazepam. The combination of fluvoxamine and proguanil can not be recommended.     

Multiple Human Cytochromes Contribute to Biotransformation of Dextromethorphan In-vitro: Role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (K_m (substrate concentration producing maximum reaction velocity) 3–13 μM). Formation of dextrorphan from 25 μM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 μm); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, ?2C19, and ?2D6 but not by those expressing CYP1A2, ?2E1 or ?3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 μM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean K_m of 259 μM. For formation of 3-methoxymorphinan from 25 μM dextromethorphan the IC50 for ketoconazole was 1.15 μM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, ?2C19, ?2D6, and ?3A4, but not by those expressing CYP1A2 or ?2E1. CYP2C19 had the highest affinity (K_m = 49 μM) whereas CYP3A4 had the lowest (K_m = 1155 μM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and ?2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.